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Daniel:Notebook/HiResChrPaint/2013-7-16
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=S3 Probe Preparation= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] Since I don't have any S3 set left, I'm going to make a new run using the various new techniques, specifically I am more confident in the dye coupling stage now. I'm going to make just the aa-dUTP (ARES labeling) style for now, since it is difficult to balance the reactions when I have a lot of samples. ==PCR Amplification== 1. Reaction Table {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" |style="background-color:#FFFF00" align="center" width="65" height="45" | | width="65" | 20 nM OligoPool | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AP1V41U | width="65" | 100 uM AP2V4 | width="65" | 100 uM AP1V61U | width="65" | 100 uM AP2V6 | width="66" | 2 mM aa-dUTP | width="65" | H20 | width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="15" | per rxn | align="center" | 0.2 | align="center" | 50 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 2.5 | align="center" | 45.7 | align="center" | 100 |- style="font-size:12pt" align="center" | height="30" | V4S3 (12.5x) | align="center" | 2.5 | align="center" | 625 | align="center" | 5 | align="center" | 5 | align="center" | 0 | align="center" | 0 | align="center" | 31 | align="center" | 581.5 | align="center" | 1250 |- style="font-size:12pt" align="center" | height="30" | V6S3 (12.5x) | align="center" | 2.5 | align="center" | 625 | align="center" | 0 | align="center" | 0 | align="center" | 5 | align="center" | 5 | align="center" | 31 | align="center" | 581.5 | align="center" | 1250 |} 2. Aliquot 100 uL into each of 12 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii vii. 72C 2 min viii. 16C hold 4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) 5. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]], added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue 6. Incubated 4 hours; resuspend in 50 uL nfH20 7. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB 8. Measure concentrations in nanodrop ===qPCR Results=== [[Image:V4S3-071613.png|600px]] ===Nanodrop Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="15" | Sample | width="65" | V4S1A | width="65" | V4S1B | width="65" | V4S1C | width="65" | V4S1D | width="65" | V6S1A | width="65" | V6S1B | width="65" | V6S1C | width="65" | V6S1D |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 97.6 | align="center" | 91.6 | align="center" | 99 | align="center" | 99.8 | align="center" | 118.6 | align="center" | 113.8 | align="center" | 117.7 | align="center" | 118.8 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 40 uL | align="center" | 3.9 | align="center" | 3.7 | align="center" | 4.0 | align="center" | 4.0 | align="center" | 4.7 | align="center" | 4.6 | align="center" | 4.7 | align="center" | 4.8 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total ug | align="center" | | align="center" | | align="center" | | align="center" | 15.5 | align="center" | | align="center" | | align="center" | | align="center" | 18.8 |} ==DpnII Digestion== 1. Prepare Samples {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="15" | Sample | width="65" | V4S3A | width="65" | V4S3B | width="65" | V4S3C | width="65" | V4S3D | width="65" | V6S3A | width="65" | V6S3B | width="65" | V6S3C | width="65" | V6S3D |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 97.6 | align="center" | 91.6 | align="center" | 99 | align="center" | 99.8 | align="center" | 118.6 | align="center" | 113.8 | align="center" | 117.7 | align="center" | 118.8 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 35 uL | align="center" | 3.4 | align="center" | 3.2 | align="center" | 3.5 | align="center" | 3.5 | align="center" | 4.2 | align="center" | 4.0 | align="center" | 4.1 | align="center" | 4.2 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | uL 10X buffer | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | uL DpnII (50U/uL) | align="center" | 2 | align="center" | 2 | align="center" | 2 | align="center" | 2 | align="center" | 2 | align="center" | 2 | align="center" | 2 | align="center" | 2 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL nfH2O | align="center" | 8 | align="center" | 8 | align="center" | 8 | align="center" | 8 | align="center" | 8 | align="center" | 8 | align="center" | 8 | align="center" | 8 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 |} 2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; [[Daniel:Notebook/HiResChrPaint/2013-7-17|Continued]] 7/17/2013]]
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